The current studies have identified genes that are expressed at a significantly different level in acute lymphoblastic leukemia cells of patients who exhibit a poor in vivo response to HDMTX. High-throughput genomic approaches to assess the expression levels of RNA transcripts in cancer cells are providing new insights into pathogenesis, classification, diagnosis, stratification, and prognosis of many human cancers [23
]. The drug resistance and gene expression profiles of leukemia cells have also been used to identify genes related to the sensitivity of ALL cells to several antileukemic agents and to forecast differences in treatment response [5
]. These findings have also revealed novel targets for the discovery of new agents to reverse drug resistance, such as our prior discovery of MCL1
overexpression in glucocorticoid-resistant ALL [5
], and the subsequent discovery that rapamycin can down-regulate MCL1
expression and increase sensitivity of leukemia cells to dexamethasone [37
]. However, prior to the current study, there has not been a comprehensive analysis of genes related to the antileukemic effects of MTX in primary leukemia cells.
We therefore evaluated MTX response in vivo after initial therapy, because this is the only possible time to assess the antileukemic effects of MTX as a single agent in patients and because there are no reliable in vitro methods. Thus, our study focused on treatment-naive ALL, and assessed de novo resistance. This revealed that WBCΔDay3
is a superior measure of in vivo MTX response when compared to the percentage drop in leukemia cells (i.e., WBC%drop
), and that WBCΔDay3
was predictive of long-term DFS. Furthermore, the difference in survival cannot simply be explained by differences in MTX systemic exposure (Figure S6
To better understand the biological basis underlying MTX response in ALL cells, we used an unbiased genome-wide approach to identify genes whose expression in primary leukemia cells in vivo was significantly related to WBCΔDay3
. This process revealed 48 genes and two cDNA clones that are highly related to the in vivo MTX response (WBCΔDay3
), even after adjusting for MTXPG accumulation (n
= 230) (Table S3
). Among those genes significantly associated with MTX response were genes involved in nucleotide metabolism (TYMS
), cell proliferation and apoptosis (BCL3, CDC20, CENPF,
), and DNA replication or repair (POLD3, RPA3, RNASEH2A, RPM1,
). The antileukemic effects of MTX involve inhibition of purine and pyrimidine synthesis, and the current findings indicate that interindividual differences in nucleotide synthesis influence the in vivo antileukemic effects of MTX. This finding was confirmed by a global test analysis that identified the nucleotide biosynthesis pathway as one of the most discriminating biological pathways related to MTX response. Significance of the global test was largely explained by three key genes (TYMS
, and CTPS
) belonging to the nucleotide biosynthesis pathway.
Our analysis also showed that low expression of DHFR, TYMS,
was significantly correlated with poor in vivo MTX response [6
]. It has been shown that DHFR, TYMS,
expression is associated with critical biological processes such as DNA synthesis and cell proliferation [43
], a finding consistent with low expression of these genes reflecting a decrease in the number of ALL cells in S-phase. As MTX selectively affects cells in the S-phase of the cell cycle [7
], it is likely that low expression of these genes explains the observed association with MTX response. To support this hypothesis, we showed that the percentage of leukemia cells in the S-phase was strongly correlated with DHFR
, and CTPS
expression and with the MTX in vivo response (Figure S5
, ). This finding does not preclude the possibility that genetically determined high TYMS
expression in ALL cells is associated with a worse prognosis in ALL, as we have previously reported [46
]. High TYMS
expression in the current study was related to higher cell proliferation, whereas higher constitutive TYMS
expression is due to a genetic polymorphism in the TYMS
promoter region. After remission is achieved, higher TYMS
expression due to the promoter polymorphism would connote a worse prognosis due to higher levels of the MTX target, thymidylate synthase, independent of cellular proliferation rates.
Our current data showed that low cell proliferation levels, in addition to our measure of in vivo MTX response, is an important ALL cell characteristic related to worse outcome. This result is in agreement with those of a previous study that found treatment-naïve blasts with a low proliferation rate are more resistant to several anticancer drugs in vitro [47
]. In the current study, the gene expression profile predicting MTX response was not associated with overall disease outcome after adjusting for other known prognostic variables in the entire study population (p
= 0.08), but was significantly related to DFS within high-risk patients (p
= 0.014). Leukemia cells of patients with high-risk ALL may intrinsically have a higher potential for poor MTX response (e.g., because of oncogenic gene fusions), in contrast to lower-risk patients whose ALL cells may acquire resistance mechanisms during the 2–3 y of therapy. Further, it is possible that patients with high-risk leukemia may be more prone to acquire resistance during therapy for various reasons (e.g., greater genetic instability in their ALL cells).
Interestingly, other known folate metabolism genes were not among the top genes, suggesting that expression of the known folate metabolism genes in pretreatment ALL cells is less important in causing de novo MTX resistance than previously thought. It may well be that these folate pathway genes are important for the acquired drug resistance that emerges during MTX treatment. It is also plausible that expression or function of these proteins is not reflected by the level of their mRNA expression in ALL cells. These possibilities merit further investigation, which is beyond the scope of the current work.
Defining the genomic determinants of ALL resistance to individual antileukemic agents is essential if the pharmacogenomics of drug resistance are to be elucidated, because the current and prior studies have shown that genes discriminating drug resistance in ALL are drug specific [5
]. To assess whether the genes we identified as related to de novo MTX resistance reflect a global resistance phenotype versus a MTX-specific effect, we compared the previously reported gene expression profiles for ALL resistance to PVAD (prednisone, vincristine, asparaginase, and daunorubicin), with the top 50 genes discriminating MTX response in the current study. This comparison revealed no overlap in the genes related to MTX resistance and the 124 genes related to prednisone, vincristine, asparaginase or daunorubicin PVAD resistance [5
]. This result indicates that genes identified in the current study are not a marker of general drug resistance or a global predictor of survival, rather they are specific to MTX (or perhaps other antifolates, but not all ALL chemotherapy). Furthermore, we applied our MTX gene expression profile to the publicly available German/Dutch dataset [5
], and documented that the MTX gene expression profile is not related to prednisolone sensitivity in this independent patient cohort (unpublished data).
Among the 50 genes that were expressed at a significantly different level in leukemia cells of MTX good responders versus poor responders, 29 were overexpressed in the MTX poor- responders. It is plausible that these overexpressed genes would be candidate targets for small molecules or other strategies to down-regulate their function, as a means to modify MTX response. Such a strategy has already proven successful in finding agents to modify the sensitivity of ALL cells to steroids [40
], and it is plausible that specific inhibitors of genes overexpressed in leukemia cells resistant to MTX could be viable targets for modulating the antileukemic effects of MTX. Likewise, strategies to invoke the expression of genes that are underexpressed in MTX poor responders could be tested for their ability to modulate MTX sensitivity.
The current study is the first, to our knowledge, to identify genes whose expression is related to in vivo MTX response in patients with newly diagnosed ALL. Our data provide new insights into the genomic basis of interpatient differences in MTX response and point to new strategies for overcoming de novo MTX resistance in childhood ALL. In addition, our data indicate that early treatment response to MTX is a significant prognostic indicator for long term DFS in children with ALL.